bioRxiv preprint doi: https://doi.org/10.1101/2020.09.29.318733; this version posted September 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. The Positive Switching RSFP Padron2 Enables Live-Cell RESOLFT Nanoscopy Without Sequential Irradiation Steps Timo Konen¹, Tim Grotjohann¹, Isabelle Jansen¹, Nickels Jensen¹, Stefan W. Hell¹, Stefan Jakobs¹,²* ¹Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany ²Department of Neurology, University of Göttingen, Göttingen, Germany *Correspondence: [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.29.318733; this version posted September 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Abstract Reversibly switchable fluorescent proteins (RSFPs) can be repeatedly transferred between a fluorescent on- and a non-fluorescent off-state in response to irradiation with light of different wavelengths. Negative switching RSFPs are switched from the on- to the off-state with the same wavelength which also excites fluorescence. Positive switching RSFPs have a reversed light response where the fluorescence excitation wavelength induces the transition from the off- to the on-state. Reversible saturable optical linear (fluorescence) transitions (RESOLFT) nanoscopy utilizes these switching states to achieve diffraction-unlimited resolution, but so far has primarily relied on negative switching RSFPs by using time sequential switching schemes. Based on the green fluorescent RSFP Padron, we engineered the positive switching RSFP Padron2. Compared to its predecessor, it can undergo 50-fold more switching cycles while displaying a contrast ratio between the on- and the off-state of more than 100:1. Because of its robust switching behavior, Padron2 supports a RESOLFT imaging scheme that entirely refrains from sequential switching as it only requires beam scanning of two spatially overlaid light distributions. Using Padron2, we demonstrate live-cell RESOLFT nanoscopy without sequential irradiation steps. Keywords: Super-resolution microscopy, Padron, switching, live cell, fluorescent protein 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.29.318733; this version posted September 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Introduction Nanoscopy, or diffraction-unlimited super-resolution fluorescence microscopy, enables the visualization of cellular structures at the nanoscale. The key to fundamentally overcome the diffraction barrier is to make adjacent molecules discernible through a fluorescence on/off state transition forcing adjacent fluorophores to emit sequentially.1 This separation of adjacent fluorophores can be implemented either in a coordinate-targeted or in a coordinate-stochastic way (for reviews see 2, 3). RESOLFT (reversible saturable optical linear (fluorescence) transitions) nanoscopy is a coordinate- targeted approach that relies on reversibly switchable fluorophores.4-7 In point-scanning RESOLFT nanoscopy, a single laser beam creating a doughnut shaped intensity distribution with a zero at its center is used to transfer molecules into a non-fluorescent off-state. Thereby, the on-state molecules and hence emission are limited to a central region smaller than the diffraction limit, which is read out by a regularly focused beam. Most implementations of RESOLFT nanoscopy rely on reversibly switchable fluorescent proteins (RSFPs), which belong to the group of GFP-like fluorescent proteins.8, 9 These proteins feature a beta-barrel structure with a central alpha-helix containing the autocatalytically formed chromophore.10 RSFPs can be reversibly toggled between a fluorescent on- and a non-fluorescent off-state by irradiation with two different wavelengths. Because RSFPs are metastable in both the on- and the off-state and the quantum yield for switching is comparatively high, the light doses and intensities required for overcoming the diffraction barrier are low compared to basically all other nanoscopy approaches.11 In fact, the light intensities used are similar to those applied in live-cell confocal fluorescence microscopy. As the intensity of irradiation is an important factor that determines phototoxicity12, 13, RESOLFT nanoscopy is particularly suitable for live-cell recordings. The switching behavior of most RSFPs has been classified as either negative or positive switching.14 Negative switching RSFPs are switched from the on- to the off-state with the same wavelength which is also used for fluorescence excitation. In positive switching RSFPs the excitation wavelength induces the off-to-on transition. In both classes of RSFPs, the respective other switching direction is triggered by a second, shorter wavelength. At present, almost all RESOLFT implementations rely on RSFPs with a negative switching mode.9 In a typical RESOLFT scheme using negative switching 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.29.318733; this version posted September 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. RSFPs, fluorophores are switched sequentially:4, 5, 11, 15-19 After initial switching to the on-state, RSFPs are switched off with a doughnut-shaped beam or a standing wave light pattern and remaining on-state fluorophores are probed with a regularly focused beam. Because for negative switching RSFPs fluorescence readout and off-switching are triggered by the same wavelength, central fluorophores are switched off during readout. As a consequence, the switching and readout sequence often needs to be repeated in order to collect enough photons if expression levels are low. This procedure is unfavorable as it increases the image acquisition time and the light dose applied to the sample. Positive switching RSFPs can be used to overcome the problem of limited fluorescence readout per switching cycle because fluorescence excitation triggers the on-switching process and hence the proteins in the center of the doughnut can be kept in the on-state for an arbitrary time during readout. Here, to achieve sub-diffraction resolution, the molecules in the periphery have to be kept in the off- state, which can be achieved by superposition of the regularly focused excitation light with the doughnut-shaped off-switching beam. In principle, the two overlaid beams can be scanned together over the sample to record a super-resolved image, without the requirement for sequential irradiation steps. For simplicity, we refer to this approach as one-step RESOLFT nanoscopy. In fact, the initial demonstration of RESOLFT nanoscopy 15 years ago was performed in the one- step mode.7 However, the utilized protein, asFP595, showed a poor switching performance and is an obligate tetramer. It is therefore not suitable as a fusion tag in live-cell imaging applications. Consequently, following realizations of the RESOLFT concept refrained from positive switching RSFPs und used negative switching RSFPs with sequential irradiation steps due to the unavailability of suitable probes. Aside from asFP595,20, 21 only few positive switching proteins have been reported so far, and none of them display the switching performance required. rsCherry,22 a red-emitting RSFP, and Padron,14 a green-emitting RSFP engineered from Dronpa,23 both display a low resistance to switching fatigue and slow switching kinetics. These characteristics were improved to some extent in Kohinoor,24 a variant based on Padron. It has been used for demonstration of point-scanning RESOLFT, but we found that Kohinoor is still prone to switching fatigue. However, resistance against switching fatigue is a key requirement for one-step RESOLFT imaging. 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.29.318733; this version posted September 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. One-step RESOLFT nanoscopy requires a positive switching RSFP which does not emit fluorescence upon irradiation with the off-switching wavelength. In addition, the vast majority of peripheral RSFPs need to reside in the off-state despite being irradiated with on-switching light, which requires the ensemble equilibrium state under simultaneous irradiation to be adjustable freely and quickly by changing the relative light intensities. If the equilibrium is reached fast relative to the beam movement during sample scanning, simultaneous irradiation with both superimposed beams alone should suffice to overcome the diffraction barrier. To engineer a positive switching RSFP with improved characteristics, we chose to rely on the well- described positive switching protein Padron.14 This RSFP displays poor expression